This invention relates to a recombinant DNA molecule comprising the adenylate cyclase toxin gene (cyaA) or a fragment thereof containing an insertion of a heterologous DNA sequence at a permissive site, wherein the fragment encodes a polypeptide exhibiting the same immunological properties as the CyaA gene product. In specific embodiments of this invention the heterologous DNA sequence encodes an immunological epitope. CyaA can be obtained from any microorganism or otherwise.
This invention also relates to a recombinant adenylate cyclase comprising a heterologous epitope at a permissive site. In specific embodiments of this invention the heterologous epitope is inserted in the N-terminal catalytic domain of the recombinant adenylate cyclase and can be presented to the immune system in association with class I major histocompatability complex (MHC). In other embodiments of this invention the heterologous epitope is inserted in the C-terminal catalytic domain and can be presented to the immune system in association with class II MHC.
This invention further relates to methods of inducing specific immune responses in animals immunized with immunological compositions comprising recombinant adenylate cyclases. In specific embodiments of this invention B cell, CD4.sup.+, and cytotoxic T cell responses are specifically induced.
With the advent of recombinant DNA techniques for modifying protein sequences, considerable work has been directed toward specifically altering or improving existing proteins. Modification of specific amino acids of enzymes of known structure to alter specificity has been achieved by site-directed mutagenesis. Although knowledge of the three-dimensional structure is restricted to a limited number of proteins, sequence comparisons between members of families of homologous proteins, as well as increasingly accurate predictions of secondary and tertiary structures, offer a useful basis for redesigning enzyme function.
Another approach of great potential interest is the insertion of new peptide sequences within a defined protein. Insertional mutagenesis has been efficiently used to study the topology of membrane proteins (Charbit et al., 1986, 1991) and, in the case of soluble proteins, to determine regions of natural flexibility (Barany, 1985a, 1985b; Freimuth and Ginseberg, 1986; Starzyk et al., 1989; Freimuth et al., 1990; Kumar and Black, 1991). Besides, insertion of a specific exogenous peptide within an enzyme could alter its catalytic or regulatory properties, thus providing a rational basis for protein engineering. Additionally, protein engineering can be used to alter or specifically direct the immune response to a defined epitope by altering the environment of the
The immune response can be divided into two distinct systems, cellular immunity and humoral immunity. Humoral immunity is mediated by soluble molecules, principally antibodies. In contrast, cellular immunity is mediated by intact cells, mostly T lymphocytes. The exact immune response generated by a foreign antigen is dependent on the nature of the antigen and the environment in which the antigen or an epitope of the antigen is presented to the immune system.
The initiating event in a humoral immune response is the binding of a B epitope to a membrane associated immunoglobulin (Ig) on a specific subset of B cells. This binding stimulates the entry of the B cells into the cell cycle, eventually resulting in the production of antibodies specifically recognizing the B epitope. The antibody response elicited by a B epitope can be T cell-dependent or T cell-independent. T cell-dependent responses are characterized by a very low primary response followed by an IgG memory response. The T cell-independent response, on the other hand, is characterized by a rapid, intense and prolonged IgM antibody response.
The cell mediated immune response is activated by T epitopes. T epitopes generally fall into two categories. Many epitopes can activate both T cells and B cells and are thus both T epitopes and B epitopes. Other T epitopes are denatured forms of native antigenic determinants and cannot activate a B cell mediated humoral immune response.
To activate a cell mediated immune response the T epitope must become associated with molecules of the major histocompatability complex (MHC). The MHC is a region of highly polymorphic genes whose products are expressed on the surface of various cells. There are two principle groups of MHC. Class I MHC is a transmembrane protein comprised of two polypeptide chains. The molecule contains an extracellular peptide binding region that is polymorphic at the peptide binding site, an immunoglobulin region, a transmembrane region, and a cytoplasmic domain that contains phosphorylation sites for a cAMP dependent protein kinase.
Class I MHC is found on virtually all nucleated cells. Class I MHC generally associate with endogenously synthesized T epitopes for presentation to the cell mediated immune system. As this association of class I MHC to specific antigen occurs in the endoplasmic reticulum, antigens that are internalized by an antigen presenting cell via the endocytotic pathway will generally not become associated with class I MHC. The association of a specific T epitope with class I MHC and incorporation of the antigen-MHC class I complex on the surface of a cell stimulates specific cytotoxic T lymphocytes (CTL). The stimulated cytotoxic T lymphocytes can then kill the cell expressing the antigen-MHC complex by granule exocytosis of a membrane pore forming protein that causes cell lysis and secretion of a cell toxin that activates DNA degrading enzymes. However, the activation of the CTL cells also requires the activation of T helper cells in certain cases.
The other major class of MHC proteins is class II MHC. Class II MHC are also transmembrane proteins. Like class I MHC, class II MHC comprises two polypeptide chains and includes a polymorphic peptide binding region, an immunoglobulin-like region, a transmembrane region and a cytoplasmic domain. However, unlike class I MHC, class II molecules are only expressed on "antigen presenting cells" such as B-lymphocytes, macrophages, dendritic cells, endothelial cells, and a few others. T-epitopes become associated with class II MHC when an antigen comprising the T epitope binds to the surface of an antigen presenting cell. The antigen enters the cell via phagocytosis or by receptor mediated endocytosis in clathrin coated vesicles. Alternatively, soluble antigens may be internalized by fluid phase pinocytosis. Once the antigen is internalized it is processed by cellular proteases in acidic vesicles resulting in peptides 10-20 amino acids long. These epitopes bind MHC class II molecules in intracellular vesicles and the complex is transported to the cell surface. The presence of the MHC class II-antigen complex on the surface of antigen presenting cells results in the stimulation of subpopulations of T helper cells. These cells aid CTL function as well as B cell responses. In addition, T helper cells can mediate inflammatory responses.
Two important factors in determining the character of an immune response are the nature of the antigen that is recognized and the intracellular or extracellular targeting of the antigen. Thus, a T cell epitope that can be targeted to enter an antigen presenting cell in a receptor mediated endocytosis dependent way will become associated with class II MHC and activate T helper cells but not CTL cells. Moreover, if a foreign T cell epitope can be directed to the cytoplasm of a target cell in a receptor mediated endocytosis independent fashion, the epitope will become associated with class I MHC and permit the activation of CTL cells. Therefore, there exists a need in the art to specifically target epitopes in order to selectively activate a cell mediated or humoral immune response.